Search

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment, and Nx-1607, is in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirmed potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing compound interaction with both the TKBD and LHR, but not the ring domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

In women globally, breast cancer is responsible for most cancer-related deaths and thus, new effective therapeutic strategies are required to treat this malignancy. Platinum-based compounds like cisplatin are widely used to treat breast cancer, however, they come with limitations such as poor solubility, adverse effects, and drug resistance. To overcome these limitations, complexes containing other platinum group metals such as palladium have been studied and some have already entered clinical trials. Here we investigated the anti-cancer activity of a palladium complex, BTC2, in MCF-7 oestrogen receptor positive (ER+) and MDA-MB-231 triple negative (TN) human breast cancer cells as well as in a human breast cancer xenograft chick embryo model. BTC2 exhibited an average IC50 value of 0.54 μM, a desirable selectivity index of >2, inhibited the migration of ER+ and TN breast cancer cells, and displayed anti-cancer stem cell activity. We demonstrate that BTC2 induced DNA double strand breaks (increased levels of γ-H2AX) and activated the p-ATM/p-CHK2 and p-p38/MAPK pathways resulting in S- and G2/M-phase cell cycle arrests. Importantly, BTC2 sensitised breast cancer cells by triggering the intrinsic (cleaved caspase 9) and extrinsic (cleaved caspase 8) apoptotic as well as necroptotic (p-RIP3 and p-MLKL) cell death pathways and inhibiting autophagy and its pro-survival role. Furthermore, in the xenograft in vivo model, BTC2 displayed limited toxicity and arrested the tumour growth of breast cancer cells over a 9-day period in a manner comparable to that of the positive control drug, paclitaxel. BTC2 thus displayed promising anti-breast cancer activity.

A series of novel mononuclear 1,3,5-triaza-7-phosphaadamantane (PTA)-based palladacycles were prepared by cleaving μ-Cl binuclear orthopalladated dimers of substituted benzylidene-2,6-diisopropylphenylamines. All complexes were fully characterized using IR and NMR spectroscopy, mass spectrometry as well as elemental analysis. In-vitro evaluation of the complexes as anti-cancer agents against the breast-cancer cell lines MCF7 and MDA-MB 231 as well the melanoma cell line ME1402 shows that four of the five complexes tested are active. These palladacycles exhibit their cytotoxicity by inducing DNA damage which subsequently triggers apoptosis. DNA binding studies using electrophoresis and spectroscopic techniques, such as UV-Vis and circular dichroism spectroscopy, confirms that the palladacycle, C2 definitely interacts with DNA. Results from these DNA binding experiments seem to rule out co-valent and intercalative binding, pointing rather to a non-covalent interaction, with electrostatic binding being the most likely possibility. It is envisioned that this would probably involve a hydrolysed or solvated derivative of C2. تم تصنيع واختبار عقار جديد مشتق من novel mononuclear 1,3,5-triaza-7-phosphaadamantane (PTA)-based palladacycles ضد مجموعة من الخلايا السرطانية. المركب اثبت فعالية باحداث الموت للخلايا.

Sarcomas are a heterogeneous group of neoplasms of mesenchymal origin. Approximately 80% arise from soft tissue and 20% originate from bone. To date more than 100 sarcoma subtypes have been identified and they vary in molecular characteristics, pathology, clinical presentation and response to treatment. While sarcomas represent

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. This study investigates the anti-cancer activity of AJ-5 in alveolar and embryonal RMS. IC50 values of ≤ 0.2 µM were determined for AJ-5 and it displayed a favourable selectivity index of >2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate, respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptosis and necrosis. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1 and a significant reduction in autophagosome flux J. Finally, pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. Together these findings suggest that AJ-5 may be an effective chemotherapeutic with a desirable mechanism of action for treating drug-resistant and advanced sarcomas.

All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate that is hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys "catalytic triad." In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzyme is maintained despite the mutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu-59) in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O(e1) in the wild-type enzyme and interacts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O(δ1.) In neither case is an atom located at the position of Glu-142 O(e2) in the wild-type enzyme. The active site cysteine of the E142L mutant was found to form a Michael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O(e2) and the substrate amino group in positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.

Recently palladium (Pd) complexes have attracted a lot of interest as chemotherapeutic agents because they have been shown to exert a significant cytotoxic effect on cancer cells. Importantly, Pd complexes have been shown to exert antitumor activity in cisplatin resistant cells and to have less side effects than cisplatin, a widely used platinum-based chemotherapeutic agent. This led to suggestions that Pd(II) compounds may have different mechanisms of action from those of cisplatin, but this is still unresolved. It is, however, generally accepted that the cytotoxic effects exerted by most metal-based compounds result from their capacity to trigger DNA double-strand breaks which activate a canonical DNA damage signaling pathway through activating ataxia telangiectasia mutated (ATM), the checkpoint kinase 2 (CHK2), and the tumor suppressor protein p53. These proteins play an important role in deciding cell fate in response to DNA damage through transactivating the cyclin-dependent kinase inhibitor p21 as well as pro-apoptotic proteins. While most chemotherapeutic agents have been described to induce cell death via apoptosis, there is increasing evidence that they can also function by initiating mitotic catastrophe and autophagy. Indeed, several studies have confirmed a complex cross-talk between apoptosis and autophagy, but while some studies indicate that autophagy inhibits the process of apoptosis, others suggest a role for autophagy in the induction of cell death. It would, however, appear that these opposing roles of autophagy depend, in part, on both the cell type and the chemotherapy used.

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 A resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4(2)32, with unit-cell parameter a = 130.49 (+/-0.05) A. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.

Cisplatin and second-generation alternatives to cisplatin, represent some of the most active and clinically useful agents in the treatment of cancer. However, their application in the clinic is marred by side-effects and drug resistance. We have identified the binuclear palladacycle, AJ-5, as a lead anticancer compound with potent activity against advanced melanomas and estrogen receptor -positive and -negative breast cancers. AJ-5 displayed an IC50 of 0.2μM in melanoma and breast cancer cell lines which was 50 fold less than that for cisplatin and our in vivo results show that it efficiently reduces melanomas in nude mice without any obvious side effects. AJ-5 also showed activity against breast cancer stem cells which are associated with drug-resistance and often not targeted by traditional chemotherapeutic agents. This study investigates the potential of AJ-5 as an anti-cancer agent in sarcomas, which are a heterogeneous group of neoplasms that account for approximately 21% of paediatric malignancies and treatment outcomes for patients with metastatic disease are dismal. Cytotoxicity assays were performed and sub-micromolar IC50 concentrations were obtained for rhabdomyosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma and osteosarcoma. Clonogenic assays show that AJ-5 greatly compromises the long-term ability of the sarcoma cells to survive and proliferate. To determine the underlying molecular mechanisms by which AJ-5 exerts its cytotoxicity, western blot analyses were performed with antibodies to key proteins involved in the DNA damage response in rhabdomyosarcoma cell lines. The results show that AJ-5 induces high levels of γH2AX, a marker of double-stranded DNA breaks, and that it may exert it's cytotoxicity through the p38 MAP kinase stress pathway. Furthermore, Annexin VFITC/propidium iodide staining, Caspase Glo assays and western blotting demonstrated that AJ-5 induce intrinsic and extrinsic apoptosis more effectively than doxorubicin, a drug currently used in the treatment of rhabdomyosarcomas. AJ-5 treatment also led to autophagy as confirmed by the formation of autophagosomes, increased levels of LC3-II and the presence of LC3 puncta. Finally, pharmacokinetic studies show that AJ-5 has a promising half-life of 11.2 hours in mice and in addition its volume of distribution is high and its clearance is low while its intraperitoneal absorption is good. Thus the PK data correlates well with our observed efficacy of the drug in our mouse model. Together these findings suggest that AJ-5 may be an effective chemotherapeutic for treating a range of drug-resistant and advanced cancers. Citation Format: Jenna Bleloch, Reyna Ballim, Angelique Blanckenberg, Selwyn Mapolie, Serah Kimani, Sharon Prince. A novel palladacycle complex with anti-cancer activity against breast cancer and melanomas also exhibits potent cytotoxicity in a range of sarcomas [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B38.

A prominent mechanism through which tumors develop resistance to DNA damaging chemotherapeutic agents is by cancer cells preferentially arresting in S-phase, thereby acquiring the ability to repair drug-induced DNA damage and consequently evading apoptosis. Combining DNA damaging therapies with targeted approaches that disrupt the DNA damage repair process may therefore prove to be more effective against such tumors. The developmentally important transcription factor TBX2 has been suggested as a novel anti-cancer drug target, as it is overexpressed in several cancers and possesses strong anti-senescence and pro-proliferative functions. Indeed, when TBX2 is silenced, we are able to reverse several features of transformation in both breast cancer and melanoma cells. We and others have previously showed that ectopic expression of TBX2 confers resistance to DNA damaging chemotherapeutic drugs, cisplatin and doxorubicin. However, whether endogenous TBX2 confers resistance to these DNA damaging agents in TBX2-driven cancers and the mechanism(s) by which this may occur have not been reported. To address this, we have silenced TBX2 in cisplatin-resistant breast cancer and melanoma cells and we show that knocking down TBX2 sensitizes the cells to cisplatin by disrupting the ATM-CHK2-p53 signalling cascade. Cell cycle analyses demonstrate that when TBX2 is knocked down there is an abrogation of an S-phase arrest but a robust G2/M arrest that correlates with a reduction in p-CHK2 and p53 levels. This prevents DNA repair resulting in TBX2 deficient cells entering mitosis with damaged DNA and consequently undergoing mitotic catastrophe. These results provide compelling evidence that targeting TBX2 in combination with chemotherapeutic drugs such as cisplatin could improve their efficacy and that it may be a viable treatment of TBX2-driven cancers. Citation Format: Serah Kimani, Sabina Wansbelen, Emily Davis, Jade Peres, Sharon Prince. The oncogenic TBX2 activates the ATM-CHK2-p53 axis to confer cisplatin resistance in breast cancer and melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B19.

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 A was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4(2)32. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily alpha-beta-beta-alpha fold. Association in the hexamer preserves the eight-layered alpha-beta-beta-alpha:alpha-beta-beta-alpha structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity.

TBX2 and TBX3 are transcription factors that play critical roles in embryonic development but have no known function in adult tissue, and their overexpression has been associated with a growing list of cancers including melanoma, sarcomas, breast, pancreatic, liver and bladder cancers (1). Previous studies in our laboratory have shown that TBX2 and TBX3 play central but different roles in oncogenesis, where TBX2 functions as a potent growth promoting factor and TBX3 contributes to tumour formation, metastasis and invasion (2-4). Additionally, our data have demonstrated that TBX2 confers resistance to cisplatin by promoting DNA repair and may also promote oncogenesis through inappropriate survival of cells with damaged DNA (5). Recent work from our group has provided strong in vitro and in vivo biological evidence that TBX2 and TBX3 are novel targets for development of anti-cancer drugs for cancers where they are overexpressed. Knocking down TBX2 by shRNA induces senescence in advanced melanoma cells, and it results in profound inhibition of proliferation of several metastatic breast cancer cell lines (2,6). Furthermore, depleting TBX2 in metastatic melanoma and cisplatin-resistant breast cancer cells leads to their sensitization to this widely used chemotherapeutic drug, suggesting that targeting TBX2 in combination with currently used therapies could improve their efficacy (5). On the other hand, when TBX3 is depleted by shRNA in some breast cancers and melanomas, cells display reduced anchorage independence, cell migration, colony forming ability and a less aggressive phenotype (2). We are in the process of exploring ways of identifying potential inhibitors of TBX2/TBX3 oncogenic activity by combining a structure-based approach that focuses on molecular recognition and interaction of TBX2/TBX3 with their target genes, and a medium throughput approach involving screening of small molecule libraries. References [1] Wansleben S, Peres J, Hare S, Goding CR, Prince S (2014) BBA - Reviews on Cancer, 1846, 380-391. [2] Peres J, Davis E, Mowla S, Bennet DC, Li JA, Wansleben S and Prince S (2010) Genes & Cancer, 1(3):272-282. [3] Mowla S, Pinnock R, Leaner VD, Goding CR, Prince S (2010) The Biochemical journal, 433(1): 145-53. [4] Peres J, Prince S (2013) Molecular cancer, 12:117.DOI: 10.1186/1476-4598-12-117. [5] Wansleben S, Davis E, Peres J, Prince S (2013) Cell Death and Disease, 4, e846; doi:10.1038/cddis.2013.365. [6] Redmond, K.L., Crawford, N.T., Farmer, H., D'Costa, Z.C., O'Brien, G.J., Buckley, N.E., Kennedy, R.D., Johnston, P.G., Harkin, D.P., and Mullan, P.B. (2010) Oncogene 29, 3252-3262. Citation Format: Serah Kimani, Deeya Ballim, Trevor Sewell, Edward Sturrock, Sharon Prince. The oncogenic transcription factors TBX2 and TBX3 as attractive targets for anticancer drug development. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C130.

The selection of point mutation at codon 164 (from isoleucine to leucine) of the dihydrofolate reductase (DHFR) enzyme in Plasmodium falciparum is associated with high sulfadoxine /pyrimethamine (SP) resistance. Using the yeast expression system that allows the detection of dhfr allele present at low level, the presence of this mutation had previously been reported between 1998–1999 in Muheza, Tanzania, an area of high SP resistance. Eighty five P. falciparum isolates, obtained from the same area between 2002 and 2003, were analysed for the presence of Leu-164 mutation, using standard protocol based on PCR-RFLP. None of the isolates had the Leu-164 mutation.